Lettuce preparation
1. Cut the lettuce leaf into pieces roughly 5 mm x 5 mm.
2. Add 15~20 lettuce pieces to 9.5 cm3 13% sorbitol solution in a test tube.
3. Incubate the tube in a water
bath at 37oC for 5
minutes. During this time the lettuce tissue
will equilibriate to the osmotic potential
of the sorbitol solution.
1. Gently stir 0.5 cm3
of the carbohydrase enzyme mixture into the
sorbitol and lettuce
preparation.
2. Return the tube to the water
bath for another 20 minutes. Gently
agitate the tube from time
to time while it is incubating. The
carbohydrase mixture contains pectinases, cellulases and
other cell wall-degrading enzymes.
Recovery of protoplasts
1. Tightly pack the spout of the filter funnel with the
nylon gauze. This will produce a fine
filter of roughly the correct pore
size (able to trap cell debris greater than 50 mm
in
diameter).
2. Pour the digested lettuce suspension into the filter funnel.
3. Wash any trapped protoplasts through the filter using
10 cm3 of 13% sorbitol
solution.
Collect all the filtrates in a centrifuge
tube. The protoplasts will pass through the filter while
the majority of the undigested cell
debris will remain on top of the gauze.
4. Ensure that the centrifuge is correctly balanced then
spin the filtrate for roughly 5 minutes at
2,000 rpm
(you may need to adjust this according to the type of centrifuge available).
5. Carefully discard the supernatant, leaving the pellet
of protoplasts at the bottom of the tube.
Use a tissue to wipe away excess liquid
from the inside wall of the tube.
6. Resuspend the pellet in approximately 0.1
cm3 of 21% sucrose
solution. This is an
osmotically balanced (isotonic) medium
in which the delicate protoplasts will not burst.
Examination of protoplasts
The protoplasts will remain stable in the sucrose solution
for several hours and can easily be seen without staining using a microscope
with a x40 objective.
Safety
Care should be exercised when loading and operating
the centrifuge. Students using the centrifuge must be closely
supervised. Spills of enzyme should be wiped up promptly using plenty of
water.